Cancer is one of the main public health problems worldwide. According to the GLOBOCAN database of the International Agency for Research on Cancer, which belongs to the World Health Organization, over 10 million cases of cancer were diagnosed worldwide in the year 2000, and the number of deaths due to cancer in the year 2000 was greater than 6 million people.
Despite all the advances that have been made in the past 20 years, cancer is still one of the main causes of death around the world. During these years successful advances have been made in the prevention and treatment of early stages of many of the different diseases that are comprised within this term. However, there have been very few advances in the development of new methods for the diagnosis or treatment of advanced and invasive stages of the disease.
During the process of becoming malignant, tumor cells change their gene expression pattern, which alters cell processes, such as cell architecture maintenance, cell adhesion, cell death and cell proliferation. These modifications generate not only changes in the cells themselves, but they also make some stromal cells receive altered molecular signals and change their behavioral pattern and gene expression pattern, acquiring a typical morphology of myofibroblasts. The molecules secreted by these myofibroblasts in response to the adjacent tumor can in turn contribute in a different manner to promoting response to the adjacent tumor can in turn contribute in a different manner to promoting tumor growth and invasion, such that a paracrine loop between the tumor and stroma is established. Scientific evidence has been generated in recent years which points with increasingly greater precision to the peritumoral stroma as one of the main promoters of tumor invasiveness as well as of therapy resistance phenomena.
The methods and products for diagnosis and therapy claimed by the authors of the present invention are within this novel research framework because they are based on the exploitation of the col11a1 gene and the proCOL11A1 protein present in stromal cells of invasive carcinomas as a potential therapeutic marker and/or target for the diagnosis, prognosis and treatment of these carcinomas.
An invasive carcinoma is a malignant neoplasm made up of epithelial cells which infiltrate and destroy the surrounding tissues; they are generally malignant tumors which, during growth, infiltrate and break the basal lamina leading to metastasis. Illustrative examples of invasive carcinomas include pancreatic cancer, renal carcinoma, transitional bladder carcinoma, bronchoalveolar carcinoma, and breast cancer, among others.
Unlike other tumors, what is surprising about pancreatic cancer is its phenotypic clinical homogeneity. The clinical behavior of pancreatic cancer is homogenous and always unfavorable, without significant differences in survival according to the stage. The number of patients with pancreatic cancer with a good prognosis is insignificant. A possible explanation is that the disease spreads even in patients with small stage I tumors. With the exception of fortuitous cases, diagnosis in an initial phase is difficult; 75% of the patients diagnosed have an advanced disease (Stages III and IV). It is a very aggressive neoplasm resistant to cytostatic treatments and only between 1 and 4% of cases are still alive five years after the diagnosis, provided that the tumor is localized and could be removed in its entirety (Warshaw A. L., and Femandes del Castillo C., N. Eng. J. Med., 1992, 326:455-465; Ahigren J. D., Semin. Oncol. 1996, 23:241-250). Pancreatic ductal adenocarcinoma was the cause of over 138,000 deaths worldwide and over 2,600 in Spain in the year 2008 (GLOBOCAN). Therefore, in the case of pancreatic cancer, both the development of early diagnosis systems and effective therapies are crucial for controlling the disease (Byungwoo R., et al., Cancer Res., 2002, 62:819-826).
In the case of renal carcinoma, 50-80% of the patients diagnosed develop metastasis, and 90-95% of these patients who have developed metastasis die during the 5 years after diagnosis (Rabinovitch R. A., et al., J. Clin. Oncol., 1994, 12:206-212; Sandock, D. S., et al., J. Urol, 1995, 154:28-31). Renal carcinoma was the cause of over 72,000 deaths worldwide and over 1,200 in Spain in the year 2008 (GLOBOCAN). Advances in the knowledge of kidney cancer genetics have allowed the classification of different types of renal tumors. The most common conventional subtype is renal cell carcinoma, which is different from the papillary, chromophobe or collecting duct subtypes. Renal oncocytoma is a benign neoplasm occasionally indistinguishable from renal carcinoma, which is a malignant neoplasm. Prior studies have demonstrated that all these histological subtypes are different both genetically and biologically, and both their morphology and behavior are determined by distinctive molecular factors (Kovacs G., et al., J. Pathol., 1997, 183:131-133), but there is still no marker that has proven to be useful in clinical trials. Treatment of renal carcinoma is extremely difficult due to its capacity to spread without producing symptoms, due to its inherent resistance to conventional systemic chemotherapy and due to the inability of radiotherapy to reduce relapse levels after nephrectomy, even in patients with node involvement or tumors not completely resected (Kjaer M., et al., Int. J. Radiat. Oncol. Biol. Phys., 1987, 13:665-672). This makes it necessary to develop alternative therapeutic approaches to treat renal carcinomas with greater efficacy.
Transitional bladder carcinoma was the cause of over 1,120,000 deaths worldwide and over 3,400 in Spain in the year 2008 (GLOBOCAN). Bladder carcinomas are scaled up from G1 to G3 according to the WHO (World Health Organization) due to the decreasing state of cell differentiation and the increasing state of aggressiveness of the disease. With respect to the stage or invasiveness, transitional cell carcinomas (TCCs) of the bladder are classified as superficial with and without involvement of the lamina propria (Ta and T1), Infiltrating carcinoma of deep layers (T2 to T4) and the rather uncommon carcinoma in situ or tumor in situ (TIS). Low grade tumors (G1) are usually confined to the mucosa or Infiltrate surface layers (stage Ta and T1). New early diagnosis systems are necessary given that 80-90% of patients in stage T2 or higher are diagnosed de novo in that highly aggressive stage and not in earlier stages (de Vere White, R. W. and Stapp, E., Oncology, 1998, 12:1717-1723). The prognosis of patients with invasive transitional cell bladder carcinoma is poor; 50% of these patients in stage T2 or higher develops distant metastases during the 2 years after diagnosis, and 69% die during the 5 years after diagnosis, even when treatment is received. Alternative therapeutic approaches are necessary for treating muscle-invasive transitional cell bladder carcinoma with greater efficiency; alternative therapeutic approaches are also necessary for treating surface tumors more efficiently than surgery, or for complementing surgery, for the purpose of preventing relapse and the tumor progression to invasiveness.
Bronchioloalveolar carcinoma (BAC), also referred to as acinar cell carcinoma, is a lung adenocarcinoma subtype with characteristics that are well enough defined to be separated from the remaining subtypes. According to the World Health Organization (WHO) classification, BAC is an adenocarcinoma in which the cylindrical tumor cells grow on the walls of a pre-existing alveolus. A key characteristic of this neoplasm is the preservation of the underlying lung parenchyma. Despite the fact that many lung adenocarcinomas have areas with a bronchioloalveolar growth pattern, the diagnosis of BAC must be restricted to those neoplasms having only this pattern. BAC are fundamentally made up of two cell types, mucinous or non-mucinous cells, the latter being the most common; many non-mucinous BAC have a central fibrous region or focal point of distortion which can simulate invasion. In most studies, BAC comprise from 1% to 5% of lung adenocarcinomas, even though some recent studies increase the incidence to 15%. 50% usually present without prior symptomatology, such as peripheral solitary nodules, which are often an Incidental finding in a chest x-ray taken for other reasons. Some BAC grow slowly for several years without spreading, however others that Initially start as solitary nodules quickly develop metastasis and bilateral spread of the disease. Adenocarcinomas having a bronchioloalveolar pattern but showing a focal point stromal invasion must be called adenocarcinomas with a bronchioloalveolar pattern; they normally show a higher degree of atypia, mainly in the focal point of invasion. Bronchioloalveolar adenocarcinomas raise different differential diagnoses with both malignant and benign processes. They can be complicated; however, one of the most relevant is ordinary lung adenocarcinoma because the survival of BAC at 5 years is greater than the former. BAC can present vascular, lymphatic and pleural invasion, however, surgery can be curative between the half and two-thirds of the cases.
Breast cancer is the neoplasm with highest rate of incidence and mortality among women, hence early diagnosis and treatment are of vital importance. In recent years, with the introduction of genetics in medicine (especially in medical oncology), various discoveries have been made that affect the prognosis and treatment of breast cancer, which have led to re-classifying this pathology in terms of its genetic profile; the most significant discoveries are related to the epithelial component of these tumors, essentially epidermal growth factors (EGFr and Her-2), which have become therapeutic targets, just like estrogen and progesterone receptors (Perou C., Sorlie T., Eisen M., 2000, Nature, 406:747-752; Nielsen T., Hsu F., Jensen K., 2004, Clin. Cancer Res, 10:5367-5374). A series of immunohistochemical markers which are very useful for differentiating between benign and malignant lesions in cases in which conventional histochemical techniques are insufficient have further been developed. In the breast, there are benign sclerosing lesions, primarily sclerosing adenosis, and the radial scar which occasionally (due to their morphological pattern) raise difficulty in differential diagnosis with malignant infiltrating lesions (such as tubular carcinoma and infiltrating ductal carcinoma); this situation is even more complex in a large-gauge needle biopsy, because since there is no complete representation of the lesion, its contours cannot be seen, which is one of the keys for the differential diagnosis, especially when myoepithelial basal cells present in benign lesions and absent in malignant infiltrating lesions cannot be recognized with standard stains.
There are immunohistochemical markers (such as p63, α-actin, smooth-muscle myosin heavy chain, calponin, s100 or CD10 protein) which stain myoepithelial cells of the breast ducts, aiding in differentiating between lesions of this type. Within these markers, α-actin, smooth-muscle myosin heavy chain and calponin are very sensitive (89%) for myoepithelial basal cells; however, they are not specific for them, also staining vascular smooth-muscle cells and stromal myofibroblasts; similar problems are raised with s100 and CD10 (Lerwill, M. F., 2004, Am J Surg Path, 28:1076-1091). Werling et al. (Werling, R. W., Hwang, H., Yaziji, H., 2003, Am J Surg Path, 27:82-90) studied a series of cases corresponding to sclerosing adenosis, infiltrating ductal carcinoma, lobular carcinoma and ductal carcinoma in situ, analyzing the reactivity pattern of the p63, smooth-muscle myosin and calponin antibodies, demonstrating that they all stained myoepithelial cells of the benign and malignant non-infiltrating cases, in turn corroborating the positively of myosin and calponin in myofibroblasts and vascular smooth-muscle cells. In that work, p63 was the marker which presented the lowest cross-reactivity with stromal fibroblasts, without staining one of them, which showed its high sensitivity and specificity for staining myoepithelial cells in comparison with the remaining antibodies studied; nevertheless, it presented some drawbacks such as the occasionally discontinuous staining of myoepithelial cells (particularly in carcinomas in situ) and, also, focal positivity in up to 11% of cases of tumor cells. Meryem et. al. in turn described positivity with p63 in 13 cases of metaplastic breast carcinoma in a total of 14 cases (Meryem, Koker, M., Kleer, C. G., 2004, Am J Surg Path, 28:1506-1512). Currently p63 is one of the most widely used markers for the differential diagnosis between benign sclerosing and malignant Infiltrating lesions of the breast; however, to date there is no stromal marker which aids in differentiating between these lesions (Mattia Barbareschi, M., Pecciarini L., Cangi G., 2001, Am J Surg Path, 25:1054-1060). There is a need for specific markers in cases in which the conventional histochemical techniques are insufficient for differentiating between benign sclerosing and malignant infiltrating lesions of the breast.
Collagen is the main component of the extracellular matrix (ECM). The correct expression of the genes encoding the different types of collagen is necessary for the correct assembly of the ECM during embryonic development and for maintenance thereof in the adult organism. Collagen XI (COL11) is a type of collagen that has been studied very little but plays a fundamental role in regulating fibril networks in cartilaginous and non-cartilaginous matrices (Li, Y., et al., Cell, 1995, 80:423-430); these fiber networks are involved in different morphogenesis processes during embryonic development in vertebrates. Transcripts of collagen XI alpha 1 chain (COL11A1) have been found during fetal development fetal in cartilaginous tissues and also in other tissues such as bone, kidney, skin, muscle, tongue, intestine, liver, ear, brain and lung (Sandberg, J. M., et al., Biochem. J., 1993, 294:595-602; Yoshioka, H., et al., Dev. Dyn., 1995, 204: 41-47). The extracellular matrix also plays an important role in certain biological processes, such as cell differentiation, proliferation and migration; therefore, the dysregulation of the expression of genes encoding the proteins making them up is associated with carcinogenic and metastatic processes (Boudreau, N., and Bissell, M. J., Curr. Opin. Cell Biol., 1998, 10:640-646; Stracke, M. L., et al., In vivo, 1994, 8:49-58). In the particular case of COL11A1, stroma fibroblasts have been proven to have high col11a1 gene expression levels in sporadic colorectal carcinomas, whereas this gene is not expressed in healthy colon (Fischer, H., et al., Carcinogenesis, 2001, 22:875-878). Col11a1 gene expression has also been associated with pancreatic, breast, colon, lung, head and neck cancer (Kim, H. et al., BMC Medical Genomics, 2010, 3:51; Iacobuzio-Donahue, C., Am. J. Pathology, 2002, 160(4):1239-1249; Ellsworth, R. E., et al., Clin. Exp. Metastasis, 2009, 26: 205-13; Feng, Y., et al., Breast Cancer Res. Treat., 2007, 103(3):319-329; J. Gast. Liv. dis., 2008; Fischer, H., et al., BMC Cancer, 2001, 1:17-18; Fischer, H., et al., Carcinogenesis, 2001, 22:875-878; Suceveanu, A. I., et al., J. Gastrointestin. Liver Dis, 2009, 18(1):33-38; Chong, I W, et al., Oncol Rep, 2006, 16(5):981-988; Whan, K., Oncogene, 2002, 21:7598-7604; Oncol Rep, 2007; Schmalbach., C. E., et al., Arch. Otolaryngol. Head Neck Surg., 2004, 130(3):295-302) and bladder cancer (WO 2005/011619), and COL11A1 protein expression has been associated with pancreatic and colon cancer (Pilarsky, C., et al., J. Cel. Mol. Med., 2008, 12(6B):2823-35; Erkan, M., et al., Mol. Cancer, 2010, 9:88-103; Bowen, K. B., et al., J. Hist. Cyt., 2008, 56(3):275-283); but COL11A1 gene or protein expression has not been associated with other types of cancer (for example, kidney cancer), nor has COL11A1 protein expression been associated with breast, lung or head and neck cancer (differential gene expression does not necessarily interfere with the differential expression of the encoding protein).
Even though there are some markers for diagnosing different types of cancer and carcinomas, there is still a need to identify new markers which allow differentiating between different types of cancers and carcinomas the prognosis of which is very different, for example, between renal carcinomas and renal benign oncocytomas, between invasive transitional bladder carcinoma and superficial transitional bladder carcinomas, between infiltrating breast adenocarcinomas and sclerosing adenosis of the breast, and between pancreatic ductal adenocarcinomas, pancreatitis and carcinomas of the ampulla of Vater (ampullary tumors), which have a much better prognosis (21-61% of patients survive 5 years after diagnosis, versus 1-5% of ductal adenocarcinomas) (Takao S., et al., 1998, Am. J. Surg., 176:467-470; Warshaw A. L. and Fernández del Castillo, 1992, N. Eng. J. Med., 326:455-465).
In turn, collagens are components of the extracellular matrix that are synthesized as procollagens. These procollagens have a central triple-helical domain with a rod-shaped structure. This domain is flanked by N- and C-terminal non-collagenous propeptides; these propeptides are removed by specific peptidases as they are secreted out of the cell, where collagen trimers are assembled to form fibers. Procollagens V and XI α1 (proCOL5A1 and proCOL11A1, respectively) are encoded by the col5a1 and col11a1 genes, respectively. They have an approximately 75% amino acid sequence homology. The VAR sub-domain in the N-terminal propeptide has the greatest sequence variation between collagens. Nevertheless, the antibodies, some of which are commercially available, which have been used until now to study the COL11A1 protein expression, are polyclonal antibodies that have been developed against the central triple-helical domain, which is highly conserved between different collagens, so they do not specifically discriminate the COL11A1 expression of other collagens with a similar amino acid sequence, the expression of which does not necessarily vary during the tumor process, so it can lead to serious errors if they are used for the development of methods and products of diagnosis and prognosis of carcinomas. This is the case for example of the anti-Col11A1 antibody marketed by Calbiochem which has a cross-reactivity with COL9 (Collagen IX) protein, which is not differentially expressed in invasive carcinomas.
Using proCOL11A1 protein as a differential marker between infiltrating squamous cell head and neck carcinoma and benign head and neck pathologies (García-Ocaña et al., Poster “Immunohistochemical validation of procollagen COL11A1 as a desmoplastic tumor stroma marker”, 18 Nov. 2010, III International Symposium of the IUOPA, Oviedo); as a differential marker between infiltrating ductal breast adenocarcinoma and sclerosing adenosis (Garcia Pravia et al., Abstract ESSR2009/218, “Anti-proCOL11A1, a new marker of infiltrating breast cancer”, British J. Surgery, 2009, 96 (S5):11; García-Ocaña et al., Poster “Immunohistochemical validation of procollagen COL11A1 as a desmoplastic tumor stroma marker”, 18 Nov. 2010, cited above); and as a differential marker between pancreatic ductal adenocarcinoma and chronic pancreatitis [García Pravia et al., Abstract ESSR2009/272, “ProCOL11A1 is an efficient marker of pancreatic cancer” British J. Surgery, 2009, 96 (S5):17; Garcia-Ocaña et al., Poster “Immunohistochemical validation of procollagen COL11A1 as a desmoplastic tumor stroma marker”, 18 Nov. 2010, cited above)], has recently been described.
Nevertheless, the antibodies available until now do not allow specifically detecting proCOL11A1 protein, so the potential use of said protein as a differential marker between different pathologies, for example, between malignant and benign pathologies, cannot be reliably put into practice to their fullest extent
Therefore, there is a need to develop antibodies which allow specifically detecting proCOL11A1. Said antibodies could be used to differentiate between different pathologies.